Tumor suppressor gene

ABSTRACT

The present invention relates to a novel tumor suppressor gene, SSeCKSS. It is based, at least in part, on the discovery of a gene, hitherto referred to as &#34;322&#34; (Lin et al., 1995, Mol. Cell. Biol. 15:2754-2762) but now referred to as SSeCKS, which was found to be down-regulated in certain transformed cells. Further, the SSeCKS gene product was subsequently shown to be a substrate of protein kinase C. SSeCKS protein has been shown to act as a mitogenic regulator and as an inhibitor of the transformed phenotype.

SPECIFICATION

The invention contained herein is based, at least in part, on research funded by NIH grant number NIH CA65787. Accordingly, the United States government may have certain rights herein.

INTRODUCTION

The present invention relates to a novel tumor suppressor gene, referred to herein as SSeCKS, its encoded protein, and methods of use thereof. It is based, at least in part, on the discovery of a SSeCKS gene which encodes a substrate of protein kinase C that functions as both a mitogenic regulator as well as a tumor suppressor.

BACKGROUND OF THE INVENTION

The inactivation of several tumor suppressor gene families (for example, those encoding p53, Rb, and APC) as a result of mutation is acknowledged to contribute to oncogenicity of several types of human cancers (Levine, 1993, Ann. Rev. Biochem. 62:623-651). Many of these so-called class I tumor suppressor genes (Lee et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:2825-2829) were identified and isolated following cumbersome pedigree and cytogenetic analyses (Sager, 1989, Science 246:1406-1412). Recently, another class of genes (class II) whose expression is known to be down-regulated in tumor cells has been shown by gene transfer techniques to encode potential tumor suppressors. These include nonmuscle α-actinin, tropomyosin I, CLP, retinoic acid receptor β₁, and interferon regulatory factor (Gluck et al., 1993, Proc. Natl. Acad Sci. U.S.A. 90:383-387; Hirada et al., 1993, Science 259:971-974; Hogel et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:985-989; Mishra et al., 1994, J. Cell. Biochem. 18 (Supp. C):171; Plasad et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7039-7043). Additional tumor suppressor gene families such as the maspin gene, rrg, and NO3 (Contente et al., 1993, Science 249:796-798; Ozaki et al., 1994, Cancer Res. 54:646-648; Zou et al., 1994, Science 263:526-529) were isolated by subtractive hybridization techniques designed to identify down-regulated genes. The ability of these genes to reverse an array of oncogenic phenotypes following gene transfer and overexpression supports the possibility for novel therapeutic modalities for cancer.

SUMMARY OF THE INVENTION

The present invention relates to a novel tumor suppressor gene, SSeCKS. It is based, at least in part, on the discovery of a gene, hitherto referred to as "322" (Lin et al., 1995, Mol. Cell. Biol. 15:2754-2762) but now referred to as SSeCKS, which was found to be down-regulated in certain transformed cells. Further, the SSeCKS gene product was subsequently shown to be a substrate of protein kinase C. SSeCKS protein has been shown to act as a mitogenic regulator and as an inhibitor of the transformed phenotype.

In various embodiments, the present invention relates to the SSeCKS gene and protein, and in particular, to rat and human SSeCKS gene and protein. Furthermore, the present invention provides for the use of such genes and proteins in diagnostic and therapeutic methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Northern blot analysis of SSeCKS RNA levels in NIH 3T3 cells versus NIH/v-src transformed cells.

FIG. 2A & B. Southern blot analysis showing that the decreased level of SSeCKS RNA in NIH/v-src cells is not due to gross deletion or translocation of the SSeCKS allele, and restriction map of SSeCKS.

FIGS. 3A-H. Nucleic acid SEQ ID NO:1 (top line, lower case letters) and deduced amino SEQ ID NO:2 acid (lower line, capital letters) sequence of rat SSeCKS cDNA.

FIG. 4A & B. Northern blot analysis showing that the transcription of SSeCKS is suppressed relatively soon after the activation of a ts-src allele (A) or the addition of fetal calf serum (FCS) to starved rodent fibroblasts (B).

FIG. 5. Northern blot analyses showing levels of SSeCKS transcripts in oncogene-transformed Rat-6 fibroblasts.

FIG. 6. Results of in vitro transcription-translation of SSeCKS cDNA.

FIG. 7A & B. Proliferation of cells overexpressing SSeCKS.

FIG. 8. "Zoo" Southern blot of SSeCKS probe to genomic DNA from various species.

FIG. 9. Northern blot analysis showing tissue-specific expression of SSeCKS in mice.

FIG. 10. Schematic diagram of SSeCKS protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to SSeCKS genes and proteins.

In one specific embodiment, the present invention relates to a purified and isolated nucleic acid molecule having the nucleic acid sequence set forth in FIG. 3, which is the rat SSeCKS cDNA. In another embodiment, the present invention relates to a purified and isolated nucleic acid molecule which hybridizes to a nucleic acid molecule having a sequence as set forth in FIG. 3 SEQ ID NO:1 under stringent hybridization conditions. This embodiment would include nucleic acid molecules from species other than rat, such as the human SSeCKS cDNA. This embodiment would also relate to genomic DNA and RNA molecules. Stringent hybridization conditions are as described in Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. In one specific, nonlimiting embodiment of the invention, stringent hybridization may be performed between DNA molecules in the Southern method, in a solution of 0.75M sodium phosphate pH 7, 1 mM EDTA, 7% SDS, 1% bovine serum albumin (BSA), and 100 microgram per ml salmon sperm DNA for 12-18 hours at 65 degrees centigrade, followed by washing twice in 50 mM sodium phosphate, 1 mM EDTA, 1% SDS, and 0.5% BSA at 65 degrees C., and twice again in the same solution without BSA at 65 degrees centigrade.

In a related embodiment, the present invention provides for a purified and isolated nucleic acid sequence which is at least 90 percent homologous to the nucleic acid molecule having a sequence as set forth in FIG. 3.

In further embodiments, the present invention provides for a purified and isolated protein having an amino acid sequence as set forth in FIG. 3 SEQ ID NO:1. The present invention also provides for a purified and isolated protein encoded by a nucleic acid molecule having the sequence set forth in FIG. 3 or (i) a nucleic acid molecule which hybridizes thereto under stringent conditions or (ii) is at least 90 percent homologous thereto.

The present invention also relates to vectors comprising the abovementioned nucleic acid molecules, including plasmid, phage, cosmid, and viral vectors. The foregoing nucleic acid molecules may be combined, in such vectors or otherwise, with nucleic acid sequences which may aid in their expression, including promoter/enhancer sequences and other sequences which aid in transcription, translation, or processing. Vectors of the invention may further comprise other sequences, such as selection markers, as used by skilled artisans.

The present invention further provides for antibodies, including monoclonal or polyclonal antibodies, directed toward the proteins of the invention, and prepared by standard techniques known in the art. It may be desirable to subject such antibodies to purification using an affinity column to which SSeCK protein is bound.

The molecules of the present invention have a number of utilities. As described in the example section below, suppression of SSeCKS expression occurs in association with transformation by certain oncogenes or by the triggering of a proliferative cycle in starved cells by the addition of serum to the growth medium. These observations indicate that SSeCKS acts as a negative regulator of mitosis. As such, the introduction of SSeCKS gene or protein into a host cell may be used to inhibit mitosis of the host cell. Introduction may be achieved either via a vector, by physical means, or by direct uptake of the SSeCKS gene or protein into the host cell.

Moreover, it has been discovered that ectopic expression of SSeCKS suppressed the ability of v-src to induce morphological transformation and anchorage-independent growth in rodent fibroblasts. Thus, the introduction of SSeCKS gene or protein into a cell may be used to inhibit the expression of a transformed phenotype by the cell.

Since many human diseases are associated with disorders of proliferation and/or with the expression of a malignant (i.e. transformed) phenotype, increasing the levels of SSeCKS DNA, mRNA, and/or protein in a patient suffering from such a disease may be beneficial. For example, the levels of SSeCKS may be increased in a malignant tumor in such a patient in order to decrease its propensity to metastasize.

Furthermore, the level of SSeCKS expression in a cell or collection of cells may be used to evaluate the mitotic state of such cells, where a low level of SSeCKS expression may bear a positive correlation with active mitosis. Furthermore, a low level of SSeCKS expression may bear a positive correlation with a malignant phenotype. Such measurements may be used in the diagnosis or staging of malignancy, or in the assessment of the effects of therapeutic interventions in a subject in need of such treatment.

EXAMPLE: CLONING AND CHARACTERIZATION OF SSeCKS

cDNAs were identified whose abundance is low in NIH 3T3 cells and decreased following the expression of the activated oncogene v-src. The transcription of one such gene, SSeCKS (pronounced "ESSEX"), was found to be suppressed at least 15-fold in src, ras, and fos-transformed cells and 3-fold in myc-transformed cells, but was unaffected in raf, mos, or neu-transformed cells. Activation of a ts-v-src temperature sensitive allele in confluent 3Y1 fibroblasts resulted in an initial increase in SSeCKS mRNA levels after 1 to 2 hours followed by a rapid decrease to suppressed levels after 4 to 8 hours. Morphological transformation was not detected until 12 hours later, indicating that the accumulation of SSeCKS transcripts is regulated by v-src and not as a consequence of transformation. Addition of fetal calf serum to starved subconfluent NIH 3T3 or 3Y1 fibroblasts resulted in a similar biphasic regulation of SSeCKS, indicating that SSeCKS transcription is responsive to mitogenic factors. Sequence analysis of a full-length SSeCKS cDNA rat clone (5.4 kb) identified a large open reading frame encoding a 148.1 kDa product, but in vitro transcription-translation from a T7 promoter resulted in a 207 kDa product. Further, sequence analysis indicated that SSeCKS has only limited homology to known genes, including the human gravin gene, where a small amount of homology exists in the 3' untranslated region. Particular data relating to these conclusions is set forth in greater detail below.

FIG. 1 depicts the results of Northern blot analysis of SSeCKS RNA levels in NIH 3T3 cells versus NIH/v-src transformed cells. A 30 microgram amount of total RNA purified by the RNAzol method from NIH 3T3 cells or NIH/v-src cells was electrophoresed through a 1% agarose-1formaldehyde gel, blotted onto Immulon N membrane, hybridized with a ₃₂ P-labelled CDNA insert containing SSeCKS sequence, washed, and autoradiographed for 3 weeks. The amount of RNA loaded was normalized by densitometric analysis of 28S and 18S RNA bands (right panel).

FIG. 2 shows that the decreased level of SSeCKS RNA in NIH/v-src cells is not due to gross deletion or translocation of the SSeCKS allele. As shown in the top panel, a 20 microgram amount of genomic DNA from NIH 3T3 or NIV v-src cells was digested to completion with EcoRI or HindIII, electrophoresed through a 0.7% agarose gel, and then blotted onto Immobilon N membrane. Fifty picogram amounts of EcoRI-cut pBluescript II KS and SSeCKS plasmid DNA were included as negative and positive controls, respectively. The blot was hybridized as described in the legend to FIG. 1, and autoradiographed for 2 days with an intensifying screen. DNA molecular size standards are shown on the left. RI refers to EcoRI, H3 refers to HindIII. The bottom panel shows the restriction map of full length SSeCKS RNA, and clone 13.2.2, isolated from a rat 3T3 library. Much of this restriction pattern is shared by both mouse and rat SSeCKS homologs, although only the rat allele contains an internal EcoRI site approximately 250 bp from the 3' cDNA terminus.

FIG. 3 depicts the nucleic acid SEQ ID NO:1 (top line, lower case letters) and deduced amino acid SEQ ID NO:2 (lower line, capital letters) sequence of rat SSeCKS cDNA. The largest open reading frame (from bases 176 to 4213) was identified using the TRANSLATE program from Genetics Computer Group (by J, Devereux, 1993, in Madison, Wis.). Glycine-rich domains in the N-terminus are underlined. Nuclear localization signals fitting th motif K(R/K)X(R/K) are boxed. A sequence consistent with a Zn finger from bases 3211 to 3280 is in boldface type. Two polyadenylation signals (AATAAA) in the 3' untranslated region are underlined.

FIG. 4 shows that the transcription of SSeCKS was suppressed relatively soon after the activation of a ts-src allele or the addition of fetal calf serum (FCS) to starved rodent fibroblasts. FIG. 4A depicts the results of experiments wherein 3Y1/ts72src cells or parental rat 3Y1 fibroblasts were grown at the nonpermissive temperature (NPT; 39.5 degrees C) for 24 hours and then shifted to the permissive temperature (PT) for v-src activity (35 degrees C). Morphological transformation was not apparent until roughly 24 hours after the temperature downshift. The level of SSeCKS RNA dropped precipitously in the transformed cells but not their untransformed counterparts. FIG. 4B shows the results of experiments in which NIH 3T3 cells and 3Y1 cells were incubated overnight with 0.25% FCS and then with 10% FCS. Total RNA isolated at various times from each cell line was analyzed for SSeCKS transcription by Northern blot analysis using ³² P-labelled SSeCKS probe. Soon after the addition of 10% FCS, the levels of SSeCKS decreased rapidly in both cell lines. The cells used for panel A were seeded at confluency at the start of the experiment whereas the cells used for panel B were subconfluent throughout the experiment.

FIG. 5 shows the results of Northern blot analyses showing levels of SSeCKS transcripts in oncogene-transformed Rat-6 fibroblasts, and demonstrates that the transcription of SSeCKS was suppressed at least 15-fold in cells transformed by src and ras and roughly 3 to 4-fold in myc-transformed cells. Each lane of the gel used to generate the blot contained 30 micrograms of total RNA from Rat-6 cells transformed with the oncogenes indicated. The rat-6 lane contains total RNA from normal control cells. The levels of SSeCKS were also found to be down-regulated 10-fold in fos-transformed cells.

FIG. 6 shows the results of in vitro transcription/translation of SSeCKS cDNA. The SSeCKS cDNA was cloned in a pBluescript II KS vector downstream of the T7 promoter, and analyzed by a coupled in vitro transcription/translation assay (TNT kit, Promega). In contrast to what was predicted, namely, a product with a molecular mass of 148.1 kDa, the 13.2.2 insert repeatedly yielded a 207 kDA product, as shown in the figure.

FIG. 7 shows the results of experiments which tested the effect of SSeCKS expression on the proliferation rates of untransformed omega packaging cells (NIH 3T3 background; panel A) or transformed cells (NIH/v-src; panel B) in the presence of serum growth factors. The SSeCKS cDNA (clone 13.2.2) was inserted into vector pBABFhygro, and transfected stably into the omega c packaging cells (panel A, solid circles). Vector alone was also transfected into these cells (open circles). Proliferation of the cells containing SSeCKS cDNA or vector alone was measured and compared (FIG. 7A). The cells were grown in media supplemented with 10% CS. FIG. 7A shows that after 4 weeks of passage, the growth rate of cells containing SSeCKS cDNA was 40% lower than that of cells containing vector alone.

Filtered supernatants from these packaging cell lines were used to infect NIH 3T3, Rat-6 and NIH/v-src--cells. Although the numbers of hygromycin resistant Rat-6 colonies arising from infection with the vector were similar to those arising from infection with SSeCKS, the initial growth rates of the colonies differed significantly. After 2 weeks, Rat-6/vector colonies were 3 to 5 mm in diameter whereas the Rat-6/SSeCKS colonies contained only 20-50 cells, indicating that SSeCKS is a negative regulator of mitogenesis.

FIG. 8 depicts the results of a Southern "Zoo" blot which measured hybridization of SSeCKS probe to DNA from a variety of species, namely genomic DNA from human (derived from HeLA cells), monkey (from CV-1 cells), rat (from Rat-6 cells), mouse (from NIH 3T3 cells), chicken (from chick embryo fibroblasts), Xenopus (from oocytes), E. coli (strain DH10), salmon sperm, and yeast cells. FIG. 8 confirms that rat and mouse 322 sequences are highly homologous. Furthermore, SSeCKS showed partial cross-hybridization to EcoRI bands from human, monkey, chicken, Xenopus, yeast, and E. coli DNA.

FIG. 9 depicts the results of Northern blot analysis of SSeCKS expression in various mouse tissues. Approximately 5.4 kb transcripts were found to be abundantly expressed in testes, with 5-10 fold lower levels in skin, brain, and lung. A 3 kb transcript was also detected in intestines, with lower levels in kidney and stomach.

FIG. 10 depicts a schematic diagram of the SSeCK protein, which contains several sequence motifs consistent with a role of transcriptional regulator, including a putative Zn finger, at least five nuclear localization signals, and several highly acidic domains typical of transactivation factors such as GAL4.

Various publications are cited herein, which are hereby incorporated by reference in their entireties.

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Ser Asn     #400     #Gly Gly Arg Lys Gly Glnsn Ser Ser Pro Asp     #                415     #Asp Ser Gly Pro Val Gluln Ala Thr Val Glu     #            430     #Val Val Pro Leu Ser Gluro Asp Val Pro Ala     #        445     #Ala Gln Gly Asn Ala Glurg Glu Lys Met Glu     #    460     #Glu Glu Leu Ser Lys Thrly Cys Val Val Ser     #480     #Asp Gly Thr Arg Ala Valer Val Ala Val Ile     #                495     #Ile Ser Ala Ser Val Thrrg Ser Pro Ser Trp     #            510     #Met Pro Pro Val Glu Gluhr Ala Gly Glu Ala     #        525     #Thr Pro Val Leu Thr Glnle Ile Ala Glu Glu     #    540     #Asp Met Val Thr Ser Gluys Asp Ala His Asp     #560     #Thr Glu Thr Ser Glu Alalu Ala Val Thr Ala     #                575     #Gly Ala Glu Glu Thr Thral Thr Glu Ala Ser     #            590     #Asp Ser Pro Asp Thr Thral Ser Gln Leu Thr     #        605     #Gly Gly Val Leu Asp Thral Gln Glu Val Glu     #    620     #Leu Gln Ala Val Ala Aspln Thr Gln Ala Ile     #640     #Thr Gln Thr Val Gln Arger Gln Val Pro Ala     #                655     #Glu Val Glu Glu Asp Sereu Glu Lys Val Glu     #            670     #Val Met Pro Lys Gly Prolu Lys Glu Lys Asp     #        685     #Gln Gly Ser Glu Thr Glyla Glu His Leu Ala     #    700     #Glu Val Thr Ala Asp Valer Leu Glu Val Pro     #720     #Leu Gln Gln Leu Met Gluys Gln Val Ile Lys     #                735     #Leu Thr Asp Ser Glu Thrlu Ser Ser Glu Thr     #            750     #Thr Ala Asp Gly Thr Glneu Ala Asp Ser Asp     #        765     #Lys Ala Thr Ala Ala Valsp Ser Gln Asp Ser     #    780     #Ala Thr Ala Gln Lys Gluhr Glu Glu Glu Ala     #800     #Ala Gln Glu Glu His Glyro Asn Asn Val Pro     #                815     #Thr Gln Gln Glu Leu Alasp Val Leu Glu Pro     #            830     #Glu Val Gly Gln Glu Glyal Trp Gln Lys Thr     #        845     #Lys Glu Glu Gln Glu Valsp Gly Glu Lys Val     #    860     #Ala Ala Asp Val Thr Tyrro Asn Ser Gln Lys     #880     #Gln Glu Lys Glu Ser Thrly Val Ala Gly Cys     #                895     #Glu Met Glu Thr Asp Valer Leu Glu Glu Gly     #            910     #Gln Val Ser Glu Glu Glylu Thr Lys Pro Glu     #        925     #Arg Asn Tyr Gly Lys Prola Pro Glu His Glu     #    940     #Arg Gly Lys Ala Leu Glyet Pro Ser Ser Glu     #960     #Gln Asp Lys Ala Gly Cysro Ser Leu Pro Asp     #                975     #Thr Val Thr Gln Thr Alaln Ser Leu Asp Thr     #            990     #Val Ile Ser Glu Thr Glyal Ile Glu Thr Val     #      10050     #Leu Pro Ala Glu Lys Seral Gly Ala His Leu     #  10205     #His Ala Glu Asp Thr Valis Trp Thr Leu Gln      025                1 - #030                1035 - #                1040     #Ile Pro Ile Ile Val Threr Gln Ala Glu Ser     #              10550     #Leu Gln Gly Glu Ile Serhr Leu His Pro Asp     #          10705     #Asp Lys Pro Asp Ala Glyrg Ser Glu Glu Glu     #      10850     #Ile Asp Lys Val Leu Lysys Glu Ser Thr Ala     #  11005     #Lys Ser Asn Lys Ile Valeu Glu Leu Glu Ser      105                1 - #110                1115 - #                1120     #Phe Ala Arg Thr Glu Thrhr Ala Val Asp Gln     #              11350     #Thr Gln Val Pro Ala Metla Tyr Asp Ser Gln     #          11505     #Trp Thr Lys Met Lys Vallu Pro Asn Arg Cys     #      11650     #Arg Glu Asp Leu Gln Valro Val Pro Gln Pro     #  11805     #Glu Met Leu Ala Ala Leula Trp Leu Ser Ser      185                1 - #190                1195 - #                1200     #Ile Glu Lys Leu Pro Proly Val Lys Val Ser     #              12150     #Asp Gly Pro Gln Leu Glnys Glu His Ala Ala     #          12305     #Asn Leu Thr Lys Glu Serlu Ala Val Ser Gly     #      12450     #Glu Arg Cys Pro Gln Lysro Lys Leu Thr Glu     #  12605     #Ser Gln Ser Lys Arg Thrys Lys Cys Leu Pro      265                1 - #270                1275 - #                1280     #Gln Arg Glu Thr Trp Glnrg Thr Cys Arg Ser     #              12950     #Ser Val Arg Pro Glu Cysal Ala His Cys Thr     #          13105     #Leu Gly Pro Trp Thr Lyssn Lys Met Leu Leu     #      13250     #Gly Arg Pro Met Ile Serrg Ser Arg Glu Gln     #  13405     -  Thr Gln      1345     __________________________________________________________________________ 

What is claimed is:
 1. An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO.1.
 2. A vector comprising the nucleic acid molecule of claim
 1. 3. An isolated host cell comprising the nucleic acid molecule of claim
 1. 